Microwave-assisted synthesis, characterization, and in vitro biological evaluation of a novel nanocomposite using molybdenum and [2,2′-bipyridine]-4,4′-dicarboxylic acid

Currently, nanocomposites are synthesized and used in various fields. One of the applications of these nanostructures is in the medical field. Therefore, the synthesis of new composites with biological properties is important. In this study, under microwave conditions, a new nanocomposite containing molybdenum and [2,2′-bipyridine]-4,4′-dicarboxylic acid (Mo/BPDA) was synthesized. The synthesized Mo/BPDA composite was subjected to biological evaluations such as antibacterial and antifungal properties by clinical and laboratory standards institute guidelines, and anticancer properties by MTT method. Characterization and structure characteristics of the Mo/BPDA nanocomposite were evaluated using XRD (X-ray diffraction pattern), FT-IR (Fourier-transform infrared), EDAX (energy-dispersive X-ray), EA (elemental analysis), TGA/DTG (thermogravimetric analysis/differential thermogravimetry), SEM (scanning electron microscopy) and BET (Brunauer–Emmett–Teller) analysis. The results indicated relatively high thermal stability (300 °C), high specific surface area (35 cm3 g−1) and uniform morphology of the synthesized Mo/BPDA nanocomposite. In antibacterial and antifungal activity, minimum inhibitory concentration (between 2 and 256 μg mL−1), minimum bactericidal concentration (between 4 and 128 μg mL−1), and minimum fungicidal concentration (between 64 and 256 μg mL−1) were tested and reported. The results showed that the antibacterial and antifungal activity of Mo/BPDA nanocomposite is higher than that of antibiotic drugs such as ampicillin, cefazolin, ketoconazole, and nystatin. In the investigation of the anticancer activity that was tested against bone cancer cells and breast cancer cells for 24 and 48 hours, cell proliferation and viability (37.3648–82.0674 tan control) and IC50 (33–43 μg mL−1) were observed. As a final result, it can be stated that the synthesized Mo/BPDA nanocomposite after the additional biological evaluations, such as in vivo study, can be used as an efficient option in treating bone cancer cells and breast cancer cells and a strong antibiotic on a wide range of infectious diseases.


Introduction
In today's world, nanotechnology and its daily use and applications are undeniable for humanity.Nano compounds have found a special place in our lives due to their unique and interesting properties.Using of nanoparticles in growth of useful plants for humans, 1 nanopackings in food, 2 nanomedicines for cancer therapy 3 and nanobers in clothes 4 are a Faculty of Pharmacy, Middle East University, Amman, 11831, Jordan b Al-Zahraa Center for Medical and Pharmaceutical Research Sciences (ZCMRS), University of Al-Zahraa for Women, Kerbala 65001, Iraq.E-mail: nather.najim@alzahraa.edu.iqsome examples.Therefore, researchers have focused on nanotechnology and are devoted to numerous researches.New nano compounds with various properties are synthesized and reported on a routine basis.Nanotechnology has made considerable advances in various elds such as agriculture, environment, basic sciences, engineering, and medicine.
Among the advances in agriculture, we can mention nanofertilizers, noble metal nanoparticles as antimicrobial agents, and protection of plants against pathogens resistant to multiple drugs. 5In this regard, we can mention gold nanoparticles, which also have environmental applications. 6,7t can be argued that the most important application of nanotechnology in the environment is the synthesis and reporting of new nano-compounds with unique abilities to absorb and remove environmental pollutants. 8Nano zerovalent irons (nZVI) used for the purication of waters and soils and the removal of dyes are some of the most important examples in this eld. 9,10Removal of azo and anthraquinone reactive dyes from industrial wastewater by magnesium oxide nanoparticles (MgO NPs) is another example of the use of nanoparticles in this eld. 113][14][15] Therefore, it can be mentioned as an example of the application of nanoparticles in organic chemistry, which is classied as basic science.In organic chemistry, synthesis, reporting, and presentation of recyclable and green catalysts have a special place. 16The synthesis and reporting of magnetic nanocatalysts is expanding in this eld.The magnet core in magnetic catalysts is Fe 2 O 3 . 17,18ano Fe 2 O 3 is also used in other elds such as the drilling uid and petroleum industry, which are examples of nanotechnology in engineering sciences. 19,20Another application of Fe 2 O 3 nanoparticles is medical diagnosis such as biosensors. 21,22esearch on nanotechnology in the eld of medicine is very extensive and many studies have been performed in many different elds of this branch of science.In various elds of medical science, such as tissue engineering, biosensing, bioimaging, 23 and applications such as drug delivery, 24 treating cancer, 25 and synthesis of nano compounds with antimicrobial, 26 antioxidant, 27 and anticancer properties, 28 it is a cornerstone for the progress of nanoscience.
Therefore, it can be stated with certainty that research in nanotechnology, including the synthesis of nano-compounds that have valuable properties, can greatly help humanity.
In the eld of medicine, nanocomposites, which are one of the most important categories of nano-compounds, have gained a good position.][35][36][37] The review of reports shows that molybdenum and nanocompounds containing molybdenum can perform many biological activities.9][40][41][42] Therefore, molybdenum can be a suitable option for synthesizing novel bioactive nanocomposites or bioactive nanocomplex using organic ligands.One of the interesting organic compounds that has the ability as ligands to synthesize complexes and nanocomposites is [2,2 0bipyridine]-4,4 0 -dicarboxylic.

Results and discussion
Mo/BPDA structure and characterization Molybdenum(VI) chloride and BPDA were subjected to the conditions mentioned in synthesis of Mo/BPDA section, including microwave radiation with a power of 350 W for 250 minutes.This radiation can create a temperature close to 120 °C (ref.52) and lead to the synthesis of a new Mo/BPDA nanocomposite.
Fig. 1 shows the structure proposed for the Mo/BPDA nanocomposite, which was conrmed by XRD, FT-IR, EDAX, and CHNO elemental analysis.The TGA, SEM and BET were other analyses that were used to characterize the Mo/BPDA nanocomposite.
As mentioned, the metal used in the synthesis of Mo/BPDA nanocomposite was molybdenum.To obtain the XRD pattern, a 100 mg Mo/BPDA nanocomposite was used.The XRD pattern of the Mo/BPDA (Fig. 2) is similar to the pattern reported previously.According to this pattern, the diffracted peaks in 2 theta angles of 8°, 10°, 24°, 50°, 70°and 75°are related to Mo crystals.4][55] Also, the BPDA showed diffraction peaks in 2 theta angles near 6°, 27°, and 29°. 56he crystal structure and the presence of molybdenum in the nanocomposite are proven (Fig. 2).The crystalline structure and nanosize of the Mo/BPDA nanocomposite (75 nm, obtained using the Debye-Scherrer equation) 57 can be attributed to the synthesis method and the use of microwave radiation. 58,59Of course, drying for 3 hours under a vacuum at 25 °C can be effective.
To obtain the SEM image, 500 mg Mo/BPDA nanocomposite was used.
The SEM image (Fig. 3(I)) of the Mo/BPDA nanocomposite is another proof of the nanostructure of the nal product and its identical morphology.
In order to determine the morphology of the Mo/BPDA nanocomposite sample with more focus, the TEM images shown in Fig. 3(II) were obtained.
Based on the obtained results, the formation of the nanocrystals with octahedral morphology was seen.These results are in agreement with the X-ray diffraction pattern that shows octahedral crystals.In addition, the morphology of the Mo/ BPDA nanocomposite is uniform, which affects the efficiency of the products.
Based on the obtained results, the formation of the nanocrystals with octahedral morphology was seen.These results are in agreement with the X-ray diffraction pattern that shows octahedral crystals.In addition, the morphology of the Mo/BPDA nanocomposite is uniform, which affects the efficiency of the products.
The FTIR spectra of BPDA and Mo/BPDA nanocomposite are shown in Fig. 4 for comparison.
In the FTIR spectra of BPDA (Fig. 4(I)) and Mo/BPDA nanocomposite (Fig. 4(II)), absorptions related to C-O (near 1100 cm −1 ), C]C (near 1400 cm −1 ), C]N (near 1500 cm −1 ), C]O (near 1650 cm −1 ) and C-H (near 2900 cm −1 ) 54,60,61 can be seen in both spectra.Only two essential differences can be seen in these two spectra, which can be used to prove the proposed structure for the nal product, as shown in Fig. 1.In the FTIR

Paper
RSC Advances spectrum of Mo/BPDA nanocomposite, the Mo-O absorption can be seen near 950 cm −1 (ref.54,62) and not observed in FTIR spectra of BPDA.Another difference is related to the O-H bond, which is observed in the FTIR spectra of BPDA (near 3300 cm −1 ) but is not present in the FTIR spectrum of Mo/BPDA.The EDAX of Mo/BPDA nanocomposite and CHNO elemental analysis of BPDA and Mo/BPDA nanocomposite are given in Fig. 5 and Table 1.
The EDAX Mo/BPDA proves the presence of carbon (C), nitrogen (N), oxygen (O), and molybdenum (MO) in its structure.
The results of Table 1 show that the total percentage of elements in BPDA is equal to 100%, but in the Mo/BPDA nanocomposite, it is about 12% less than 100%.This difference can be attributed to the presence of molybdenum in the Mo/BPDA nanocomposite.In addition, the comparison of the percentage of elements in BPDA and Mo/BPDA nanocomposite shows that the percentage of elements in BPDA is lower compared to that in the Mo/BPDA nanocomposite; for example, the percentage of carbon in BPDA was 59% and in Mo/BPDA nanocomposite it was 52%.
The results of N 2 adsorption/desorption measurements on the synthesized Mo/BPDA nanocomposite (Fig. 6) indicated its high specic surface area.
As shown in Fig. 6, the adsorption/desorption isotherm of the Mo/BPDA nanocomposite was type III.Table 2 shows the Brunauer-Emmett-Teller (BET), Barett-Joyner-Halenda (BJH) pore volume and mean pore diameter (MPD) of the Mo/BPDA nanocomposite.A high specic surface area and high porosity are the factors that lead to increasing the applications and performance of nanoparticles.As discussed in 3-2, the results of these factors and high performance were fully observed in biological assays.
These parameters also depend on the synthesis method, 63,64 and the high results obtained in this study also indicate the appropriateness of the synthetic method used.Thermal stability, which was performed using TGA/DTG (Fig. 7), was another study performed to characterize the Mo/ BPDA nanocomposite.
The TGA/DTG curve of Mo/BPDA nanocomposite showed two specic weight loss.One of which can be attributed to the decomposition of BPDA (near 300 °C) and the other to the destruction of the complex network (350-600 °C).According to the TGA/DTG curve, Mo/BPDA nanocomposite is stable up to 295 °C, a relatively good temperature.Therefore, the synthesized Mo/BPDA nanocomposite has acceptable thermal stability.
To compare the evaluation of antimicrobial activities, tests were performed on molybdenum(VI) chloride, BPDA, and Mo/ BPDA, as well as commercial drugs (ampicillin and cefazolin).The results obtained from the tests are given in Table 3.
The MIC results obtained from the antimicrobial tests of the nal product against Edwardsiella tarda, Klebsiella pneumoniae, Bacillus cereus, Shigella dysenteriae, Streptococcus iniae, Rhodococcus equi at 8, 2, 64, 32, 32 and 4 mg mL −1 , respectively.The MBC results were 16, 4, 128, 32, 64 and 16 mg mL −1 , respectively.For example, MBC of several concentrations of Mo/BPDA against Klebsiella pneumoniae are shown in Fig. 8. Comparing the results between the raw materials (molybdenum(VI) chloride and BPDA) with the nal product (Mo/ BPDA), it was observed that the best effectiveness is related to the Mo/BPDA.The obtained result can be attributed to some physical parameters of the nal product, such as the nano-size structure and its high specic surface area.Therefore, with the increase of the specic surface area, the contact of the nal product with the studied species increases, and, as a result, better effectiveness is achieved. 65,66][40][41][42] The noteworthy point in the results is that ampicillin is not effective against Edwardsiella tarda, Klebsiella pneumoniae, Bacillus cereus, and Streptococcus iniae, and cefazolin is not effective against Bacillus cereus, Shigella dysenteriae, and Rhodococcus equi, but Mo/BPDA is remarkably effective.Therefore, the Mo/BPDA can be a suitable antimicrobial candidate and, as observed, it showed better effectiveness in competition with ampicillin and cefazolin.
In antifungal evaluations to compare the assessment of antimicrobial activities, tests were performed on molybdenum(VI) chloride, BPDA, and Mo/BPDA as well as commercial drugs (Ketoconazole and Nystatin).The results obtained from the tests are given in Table 4.
The MIC results obtained from the antimicrobial tests of the nal product against Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus Fresenius, and Fusarium oxysporum were 256, 128, 64, and 64 mg mL −1 , respectively.The MFC results were 256, 256, 64, and 128 mg mL −1 , respectively.For example, the MFC of several concentrations of Mo/BPDA against Aspergillus fumigatus are shown in Fig. 9.
Here is also a comparison of the results between the raw materials (molybdenum(VI) chloride and BPDA) with the nal product (Mo/BPDA), it was observed that the best effectiveness is related to Mo/BPDA.
The proposed result of the physical parameters of Mo/BPDA in the high antibacterial activity compared to the raw materials can also be stated in the antifungal activity. 65,66he comparison of Mo/BPDA with drugs also proved that ketoconazole is not effective against Cryptococcus neoformans, and nystatin is not effective against candida albicans, but Mo/ BPDA showed good effectiveness.Therefore, regarding antifungal activity, it can be stated that Mo/BPDA can be introduced as a suitable candidate with acceptable antifungal activity.Anticancer activity of Mo/BPDA nanocomposite.The anticancer activity of the nal product (with concentrations of 6.25 mg mL −1 , 12.5 mg mL −1 , 25 mg mL −1 , and 50 mg mL −1 ) against bone cancer cells (MG-63 -CRL-1427) and breast cancer cells (MCF7 -HTB-22) at 24 h and 48 h were tested (Fig. 10).
In the evaluations, the IC 50 value was calculated and Fig. 8 shows the obtained line equation that was used to calculate IC 50 .
The results of the anticancer activity including cell proliferation and viability then the control in different concentrations and temperatures and IC 50 are given in Table 5.
The results showed that 50 mg mL −1 of Mo/BPDA was the most effective concentration against cancer cells, and 48 hours was the optimal time.In these conditions, the best cell proliferation and viability than the control for bone cancer cells was 37.3648 mg mL −1 and for breast cancer cells 39.2790 mg mL −1 .
The IC 50 for bone cancer cells at 24 h and 48 h were calculated as 39 mg mL −1 and 33 mg mL −1 , respectively, and for breast cancer cells at 24 h and 48 h were calculated as 43 mg mL −1 and 35 mg mL −1 , respectively.
Statistical studies of IC 50 at 24 h and 48 h were carried out and the calculated P-value are given in Table 6.
The results proved that the concentration of the nal product is the critical parameter at 24 and 48 hours.
Therefore, based on the results obtained from cell proliferation and viability than control, IC 50 , and statistical studies, it can be stated that with the increase in the concentration and time, the nal product is more in contact with the cells and leads to their destruction in a signicant amount.In anticancer activity as well as antibacterial and antifungal activity studies, the physical properties of the nal product that lead to an increase in the contact surface, i.e. being nano-sized and having a high specic are surface area, being deduced. 65,66[40][41][42]

Materials and characterization devices
Chemical materials and biological materials.The American Type Culture Collection and Sigma-Aldrich were used to prepare chemicals and bacterial strains.American Type Culture Collection was also the source of the studied cancer cells.
Equipment and characterization devices.The equipment and characterization devices used in the analysis of this study are given in Table 7.

Biological evaluation
Antibacterial investigation of Mo/BPDA.The Clinical and Laboratory Standards Institute's standards were followed for measuring minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) when investigating the antibacterial activities of Mo/BPDA nanocomposite.The concentrations of the synthesized Mo/BPDA nanocomposites were between 1 mg mL −1 and 2048 mg mL −1 .The tests were carried out on 1 × 10 5 CFU mL −1 concentration of the studied strains.For the MIC study, 90 mL of the Mueller-Hinton broth, 10 mL of bacterial strain, and 100 mL microliters of Mo/BPDA nanocomposites (a separate concentration in each plate well) were poured into 96 well plates and shaken for 48 h at 37 °C in an incubator.The lowest concentration at which the mixture was clear and had no turbidity was reported as MIC.For the MBC study, the contents of the 96-well plate used in MIC were cultured separately on Mueller-Hinton agar and incubated for 72 h at 37 °C.The lowest concentration in which bacteria did not grow was reported as MBC. 68,69ntifungal investigation of Mo/BPDA.The Clinical and Laboratory Standards Institute's standards were followed for measuring minimum inhibitory concentration (MIC), and minimum fungicidal concentration (MFC) when investigating the antifungal activities of Mo/BPDA nanocomposite.Concentrations of the synthesized Mo/BPDA nanocomposites were between 1 mg Paper RSC Advances mL −1 and 2048 mg mL −1 .The tests were carried out on 1 × 10 5 CFU mL −1 concentration of the studied strains.For the MIC study, 90 mL of the Sabouraud-Dextrose broth, 10 mL of fungi strain, and 100 mL microliters of Mo/BPDA nanocomposites (a separate concentration in each plate well) were poured into 96 well plates and shaken for 48 h at 27 °C in an incubator.The lowest concentration at which the mixture was clear and had no turbidity was reported as MIC.For the MFC study, the contents of the 96-well plate used in MIC were cultured separately on Sabouraud-Dextrose agar and incubated for 72 h at 27 °C.The lowest concentration in which fungi did not grow was reported as MFC. 68,69nticancer investigation of Mo/BPDA.The measurement of the cell proliferation and viability of control, and IC 50 was carried out and reported based on the MTT methods in the investigation of the anticancer activities of the Mo/BPDA nanocomposite.At concentrations of 6.25 mg mL −1 to 50 mg mL −1 , synthesized Mo/BPDA nanocomposites were tested and studied.The tests were carried out at 24 and 48 hours on bone cancer cells (MG-63 -CRL-1427) and breast cancer cells (MCF7 -HTB-22). 70In the evaluations, the passage number of cells was 3 and the concentration of cells was 1.2 × 10 4 cells per well.

Conclusions
The new nanocomposite was synthesized using molybdenum(VI) chloride and BPDA under microwave conditions.XRD, FT-IR, EDAX, CHNO elemental analysis, TGA, SEM, and BET were used to identify and conrm the structure of the newly synthesized Mo/BPDA nanocomposite.The antibacterial, antifungal, and anticancer activities of the synthesized Mo/BPDA nanocomposite were tested.MIC, MBC in antimicrobial activity and IC 50 in anticancer activity were tested and reported.In the anticancer activity, investigations were carried out on bone cancer cells and breast cancer cells, and IC 50 was 33 mg mL −1 for the bone cancer cells and 35 mg mL −1 for the breast cancer cells.Regarding antibacterial and antifungal activity, the effectiveness of the synthesized Mo/BPDA nanocomposite on some of the studied strains was higher than that of ampicillin, cefazolin, ketoconazole, and nystatin.The high efficacy of the synthesized Mo/BPDA nanocomposite can be attributed to some of its unique physical and chemical characteristics, such as the presence of bioactive compounds in its structure, as well as its nanosize and high specic surface area.The newly synthesized Mo/BPDA nanocomposite is capable of being introduced as a highly bioactive candidate with antibacterial, antifungal, and anticancer properties as a nal result.

Table 1
EA of the Mo/BPDA nanocomposite

Table 3
Antibacterial activity of the Mo/BPDA nanocomposite a a MIC and MBC value: mg mL −1 .

Table 5
Anticancer results of the Mo/BPDA nanocomposite

Table 7
Analysis and characterization using different devices